High‐Throughput Proteomics on Antibody‐based Microarrays: the Importance of Probe and Surface Design

Steinhauer, C.; Wingren, C.; Borrebaeck, C. A. K.

In analogy to DNA microarrays, protein microarrays offer a new distinct possibility to perform sensitive high‐throughput global proteome analysis. However, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. The analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. We have recently generated a human recombinant single‐chain Fv antibody library, genetically constructed around one framework, the nCoDeR‐library, containing 2 × 1010 clones. Single framework antibody fragments (sinFabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). However, the choice of framework is critical. We have shown that the selected nCoDeR framework displayed excellent functional on‐chip stability and arrayed dehydrated probes retained their activity for several months. Furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. An in‐house‐designed substrate, macroporous silicon coated with nitrocellulose (MAP3‐NC7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. We have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. Using a novel affinity tag, the double‐(his)6‐tag, we increased the binding efficiency of sinFab‐molecules to Ni2(+)‐coated solid supports, thereby allowing nonpurified probes to be directly applied.